Hyperinsulinemia has been strongly implicated as a risk factor for the accelerated development of vascular disease and is associated with both type 1 and type 2 diabetes mellitus. A mechanism for this action of insulin has not been found. We propose that insulin stimulates the pro- duction, secretion and action of the vasoactive peptide, endothelin, which itself is felt to importantly contribute to several forms of vascular disease. In these studies, we will determine the mechanisms by which insulin (in the presence and absence of hyperglycemia) regulates ET-1 gene transcription. Using gel mobility shift and DNase footprinting, we will identify the specific sequences in the ET-1 promoter and the inducible nuclear protein necessary for insulin action. To provide functional data that these promoter elements are necessary for insulin-induced ET-1 transcription, we will carry out ET-1 promoter/CAT gene reporter studies. We will determine the effects of insulin/glucose, on endothelin secretion from cultured aortic endothelial cells, and in diabetic or hyperinsulinemic rats. The latter in-vivo studies should confirm the in-vitro findings. To examine the effects of insulin and glucose on endothelin receptors, we will carry out binding studies on vascular smooth muscle cells (VSMC) from diabetic or hyperinsulinemic rats, analyzed by scatchard analysis. We will also determine the effects of these treatments on ETA (vascular) receptor gene expression. Our initial studies indicate that the in-vivo milieu induces significant changes in ET receptor numbers from short term cultured VSMC. Finally, we will show that, 1) insulin augments the mitogenic action of endothelin on VSMC through the enhancement of ET-1 stimulation of protein kinase C or intracellular calcium mobilization; and 2) that the in-vitro mitogenic action of insulin is a direct result of ET secretion and action. The results of our studies will provide a theoretical basis for the proposal that hyperinsulinemic induced ET-1 secretion or action is a risk factor for accelerated vascular disease.